Propylene glycol-based antifreeze as an effective preservative for DNA metabarcoding of benthic arthropods

Maintaining the integrity of DNA in bulk environmental samples from source to laboratory is crucial for capturing the true range of taxa present within an ecosystem. Preservation consideration of DNA is particularly important if samples are being collected in remote areas and by non-specialist citizen scientists in nationwide programs. Traditionally, absolute ethanol is used as the preferred preservative for environmental samples collected for downstream DNA metabarcoding analyses. However, transport, shipping and DNA extraction of samples preserved in ethanol is a lengthy procedure due to safety restrictions and the requirement of full ethanol evaporation prior to extraction. We examined the efficacy of an easily accessible, non-toxic propylene glycol-based antifreeze as an alternative to absolute ethanol for preserving macroinvertebrate DNA from bulk-benthos DNA samples. We tested the differences in both cytochrome oxidase I (COI) exact sequence variants (ESVs) and COI taxonomic orders detected in both ethanol and antifreeze samples using two processing methods (no evaporation of preservative versus full evaporation). In addition, we assessed the detection of families and genera within the Arthropoda phylum for preservative type, site and processing method. Our results suggest that antifreeze is a suitable alternative to ethanol, a greater global ESV richness reported for antifreeze samples. Additionally, a higher proportion of arthropod reads in ESVs were detected in antifreeze (average 69%) compared with ethanol (average 53%). Finally, antifreeze samples produced similar results for the different processing methods, whereas ethanol samples failed to produce similar results without prior evaporation. Although ethanol is currently widely used for DNA preservation, our results demonstrate that by using antifreeze, it is possible to achieve similar taxonomic coverage and community assemblages of bulk-benthos DNA samples for macroinvertebrates, with the added simplicity and shorter laboratory processing time achieved using an easily available, unregulated preservative.